ABSTRACT: The long term objective and goal of this proposal throughout its tenure has been elucidation of factors controlling SR calcium flux: 1) the mechanism by of energy transduction that allows the energy from high energy phosphate to support translocation of calcium ion across SR phospholipid bilayer and 2) the potential role of cyclic nucleotide related factors in calcium in the SR. Based on our recent data, we will focus on the following issues: 1) We will examine factors which influence the ATP hydrolysis cycle and the partial reactions thereof and study their relationship to calcium flux. Specifically, we will examine the hypothesis that calcium gradients may influence the direction of the enzyme cycle by facilitating the binding of calcium to low energy forms of acyl phosphate and that this effect may be altered by magnesium. The direction of flux and the energy transients (low energy; high energy) may thus be explained based on fundamental affinity kinetics. this model will be further investigated by examining the interaction of calcium and magnesium in loaded and unloaded sarcoplasmic reticulum and their relationship to high energy and low energy acyl phosphate species. Specific probes of nucleotide binding sites and heavy metal analogues of calcium and magnesium will serve to further refine the model. Membrane fluidity assessment and perturbents will be utilized to further expose the nature of the lipid:protein interaction in the cycle interaction. The relationship of cycle mechanism to anion co-transport will be examined to delineate the relationship of the latter to Ca2+ transport kinetics. 2) We propose to identify the specific G-proteins in SR which modulate our observed effects on the calcium release channel exposed to GTP analogues. We will do this by attempting to identify the various species of G-proteins affinity labelled with GTP, labelled with ADP ribose by NADP and cholera toxin or pertussis toxin, or identified by specific antibodies. We will examine the functional specificity of these G-proteins by adding the activated (alpha) subunit to calcium channels inserted into membrane bilayers. In addition to comparing the different available G-protein, we will contrast the effects of these proteins on release channels of cardiac and skeletal muscle SR functionally (in lipid bilayers) biochemically and constitutionally using both SR vesicles and purified release proteins.